pggn jk United States Prevention, Pesticides EPA 640-C-09-003
C, Environmental Protection and Toxic Substances October 2009
l#crn Agency ^
Endocrine Disruptor
Screening Program
Test Guidelines
OPPTS 890.1550:
Steroidogenesis
(Human Cell Line -
H295R)
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NOTICE
This guideline is one of a series of test guidelines established by the Office of
Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection
Agency for use in testing pesticides and chemical substances to develop data for submission to
the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, etseq.), and section
408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
The OPPTS test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test,
and is also used by EPA, the public, and the companies that are subject to data submission
requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these
guidelines are not binding on either EPA or any outside parties, and the EPA may depart from
the guidelines where circumstances warrant and without prior notice. The procedures contained
in this guideline are strongly recommended for generating the data that are the subject of the
guideline, but EPA recognizes that departures may be appropriate in specific situations. You
may propose alternatives to the recommendations described in these guidelines, and the
Agency will assess them for appropriateness on a case-by-case basis.
For additional information about OPPTS harmonized test guidelines and to access the
guidelines electronically, please go to http://www.epa.gov/oppts and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150
through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OPPTS 890.1550: Steroidogenesis (Human Cell Line - H295R)
(a) Scope.
(1) Applicability. This guideline is intended to meet testing requirements of
the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C.
136, et seq.), and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21
U.S.C. 346a).
(2) Background. The Endocrine Disruptor Screening Program (EDSP)
reflects a two-tiered approach to implement the statutory testing
requirements of FFDCA section 408(p) (21 U.S.C. 346a). In general, EPA
intends to use the data collected under the EDSP, along with other
information, to determine if a pesticide chemical, or other substances, may
pose a risk to human health or the environment due to disruption of the
endocrine system.
This test guideline is intended to be used in conjunction with other
guidelines in the OPPTS 890 series that make up the full screening
battery under the EDSP to identify substances that have the potential to
interact with the estrogen, androgen, or thyroid hormone (Tier 1
"screening"). The determination will be made on a weight-of-evidence
basis taking into account data from the Tier 1 assays and other
scientifically relevant information available. The fact that a substance may
interact with a hormone system, however, does not mean that when the
substance is used, it will cause adverse effects in humans or ecological
systems.
Chemicals that go through Tier 1 screening and are found to have the
potential to interact with the estrogen, androgen, or thyroid hormone
systems will proceed to the next stage of the EDSP where EPA will
determine which, if any, of the Tier 2 tests are necessary based on the
available data. Tier 2 testing is designed to identify any adverse
endocrine-related effects caused by the substance, and establish a
quantitative relationship between the dose and that endocrine effect.
(3) Source. This protocol covers the plating of H295R cells (ATCC CLR-
2128) in a 24-well plate setup and exposure of the cells to test chemicals.
It provides detailed information regarding the handling of samples
including the extraction of hormones from medium, storage of samples
and extracts, and references detailed SOPs or protocols for the analysis of
testosterone (T) and 17beta-estradiol (E2). The protocol also describes
the procedure for the Live/Death® cytotoxicity test to evaluate any
possible effects of the test chemicals on cell viability.
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The protocol provides a complete list of required reagents and solvents,
and describes the preparation of all solutions and reagents used during
the exposure experiments with the cells as well as during the subsequent
extraction, hormone analyses and cell viability measurements. It also
provides descriptions of QA/QC procedures that must be performed prior
to initiating chemical testing and the analysis of positive controls with each
run to verify proper performance of the cells.
Cell culturing procedures (including initiating cell cultures from frozen
stock, cultivation and splitting of the growing cells, the freezing of cells for
storage in liquid nitrogen and the maintenance of a proper cell culture
diagram to track the progress of a cell line) are described in Section (f).
Purpose. The H295R Steroidogenesis Assay is intended to identify xenobiotics
that affect the steroidogenic pathway beginning with the sequence of reactions
occurring after the gonatotropin hormone receptors (FSHR and LHR) through the
production of testosterone and estradiol/estrone. The steroidogenic assay is not
intended to identify substances that affect steroidogenesis due to effects on the
hypothalamus or pituitary gland.
Important Considerations.
~ Due to changes in the estradiol producing capacities of the cells with
passage/age (Hecker et al. 2006), cells are cultured following a specific
protocol before they can be used in experiments: After initiation of an
H295R culture from an original ATCC batch following the procedures
outlined below (Subsection (f)(3) Starting Cells from Frozen Stock), cells
need to be grown for five (5.0) passages {i.e., cells need to be split 4-times).
Passage five cells are then frozen in liquid nitrogen (Subsection (f)(5)
Freezing H295R Cells) for storage. For cells started from these frozen
batches, the procedures as described in Subsection (f)(3) (Starting Cells
from Frozen Stock) are followed. These cells are cultured for a minimum of
four (4) additional passages (passage # 4.5) prior to their use in exposure
experiments. The maximum number of passages that provide stable results
is 10.
~ Prior to initiation of cell cultures, it is recommended that Nu-serum be
analyzed for background testosterone and estradiol concentrations.
~ Prior to freezing cells for later use, a subset of passage five (5.0) cells is run
in an QC plate (Subsection (g)(2)(iv) Quality Control Test for Cell
Performance) to verify whether basal production of hormones and response
to a positive control chemical meet the quality criteria in Table 5.
Laboratories that have already frozen passage five (5.0) cells can thaw one
of the frozen batches, grow it for three (3) passages (passage # 3.5), and
use these cells for the QC plate run.
~ All procedures involving cells are conducted using great care and the
general considerations listed below should be followed.
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• ALWAYS perform all operations with cells in a GENTLE manner.
• ALWAYS remove media/reagents from the plate well borders.
• ALWAYS add media/reagents by resting the pipette against the well
wall.
• NEVER vortex or vigorously shake cells.
~ To avoid clumping of cells in suspension that can result in potential seeding
differences among wells of a plate, cells should be always thoroughly
resuspended after trypsinization and centrifugation by gentle aspiration
using a pipette.
~ Prior to the initiation of cell culture and any subsequent experiments each
laboratory is expected to
• demonstrate that the hormone measurement system to be used can
detect hormone concentrations in supplemented medium with
sufficient accuracy and precision to meet the QC criteria specified in
Table 1, and
• conduct a qualifying experiment demonstrating that the laboratory is
capable of maintaining and achieving appropriate cell culture and
experimental conditions required for chemical testing (Subsection
(9)(2)(iii)).
~ Sufficient basal hormone production depends on a number of different
factors including seeding density and cell passage. However, in some
cases basal E2 production may be still less than desired (< 40 pg/mL)
regardless of cell density and age/generation. In such cases 22-R
Hydroxycholesterol can be added to the supplemented medium at
concentrations between 20 and 40 |j,M to increase basal production of E2.
In any case, performance of the assay must be demonstrated in the
proficiency test (Subsection (g)(2)(iii)) prior to the conduct of any
experiment.
~ Prior to initiation of the actual exposure experiments, it is recommended that
each chemical be tested for potential interference with the hormone
measurement system to be utilized. This is of particular relevance for
antibody-based assays such as ELISAs and RIAs because some chemicals
can interfere with these tests (Shapiro and Page 1976).
~ It is critical that exact volumes of solutions and samples are delivered into
the wells during dosing because these volumes determine the
concentrations used in the calculations of assay results. Therefore, only
pipettes of the appropriate volume range are used (e.g., use a 0.1 - 1 |jL
pipette for pipetting 1 |jL of test solution stock into wells; use a 1 mL pipette
for pipetting 0.99 mL of medium into wells) to avoid introduction of errors
due to imprecise pipetting.
~ Although the H295R cells are a human cell line and are considered non-
infectious, it is recommended that proper biosafety measures be observed
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such as the use of a biosafety cabinet, proper disposal/autoclaving of waste,
and sterilization of all surfaces prior to and after work with the cells. Gloves
and lab coat are considered to be the minimal amount of protection to be
worn. Avoid wearing gloves that could trap liquid nitrogen next to the skin.
Snug fitting non-permeable (waterproof) gloves are best. Eye protection is
also recommended when exposure to splashing liquid nitrogen, solvents
and biological material is possible. Special safety requirements need to be
considered when working with ether, tritium and ethidium bromide.
Definitions and Acronyms.
DMEM w/Hams F-12: Dulbecco's Modified Eagles Medium with Nutrient
Mixture F-12 Hams.
Passage: Identifier that describes the number of times cells were split after
initiation of a culture from frozen stock. The initial passage that was
started from the frozen cell batch is assigned the number one (1).
Cells that were split 1x are labeled passage 2, etc.
PBS: Dulbecco's Phosphate Buffered Saline Solution.
QC: Quality Control.
QA: Quality Assurance.
SOP: Standard Operating Procedure.
Preparation of Solutions and Reagents for Cell Culture.
(1) Dulbecco's Phosphate Buffered Saline (PBS).
(i) Equipment, Materials and Reagents.
Equipment: Stir Plate; Analytical Balance; Pump for Filtration
Materials: Weigh Boats; Chemical Spatula; Pasteur pipettes; Stir Plate;
1000 ml Graduated Cylinder; 1000 ml Autoclaved Amber
Bottle; 500 ml Bottle Top Filter (0.22 micron Cellulose Acetate;
Low Protein Binding Membrane; Sterilizing; Corning Inc Cat#
430513); 10L Carboy or Storage Container.
Reagents:
~ KCI (potassium chloride); J.T. Baker Cat# 3040-01
~ KH2PO4 - monobasic, 99% (potassium phosphate); Aldrich
Cat# 22130-9
~ Na2P04 - 99+% (sodium phosphate); Aldrich Cat# 21988-6
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~ NaCI - 100.2% (sodium chloride); J.T Baker Cat# 3624-05
Method, Procedures and Requirements.
1. Measure out 9L of ultra clean grade water (e.g., nanopure)
into a carboy or storage container.
Note: As an alternative to the 10 L volume described here,
smaller amounts of PBS buffer can be made with
appropriate adjustments to the amounts of reagents used.
2. Place the carboy on a stir plate and place the stir bar in the
carboy. Turn on the stir plate.
3. Add 2.0 g of KCI, 2.0 g of KH2P04, 11.5 g of Na2P04, and 80
g of NaCI to the carboy.
Note: Add the chemicals slowly and wait for them to dissolve
before proceeding.
Note: Remember to rinse any traces of chemical out of the
weigh boat and into the solution using nanopure water.
4. Bring the total volume up to 10L with ultra clean grade water.
5. Adjust the pH to 7.4 using 10M HCI (if the pH needs to be
decreased) or 10M NaOH (if the pH needs to be increased).
Note: Add the HCI or NaOH one drop at a time (using a
Pasteur pipette) as the high concentration will change the pH
considerably.
6. Prior to use in the cell culture, sterilize the buffer by
autoclaving or filtration using a sterile 0.22 micron pore bottle
top filter in a 1L amber autoclaved bottle.
7. Label the bottle as follows:
• PBS
• H295R Cells
• "Filter Sterilized" (only for the work solution to be used
with cells)
• pH = 7.4
• Preparer's initials
• Expiration date
8. Store the buffer at room temperature.
9. PBS can be used for up to 6 months, after that new PBS
buffer should be made.
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Note: Alternatively, Dulbecco's Phosphate Buffered
Saline 10x, Modified, Without Calcium Chloride and
Magnesium Chloride, Liquid, Sterile-Filtered, Cell Culture
Tested can be Purchased from Sigma (Cat # D-1408).
Stock and Supplemented Medium.
(i) Equipment, Materials and Reagents.
Equipment: Biosafety Cabinet; Stir Plate; Analytical Balance; Pump for
Filtration.
Materials: Weigh Boats; Chemical Spatula; Pasteur pipettes; Stir Plate;
1000 ml Graduated Cylinder; 1000 ml Autoclaved Amber
Bottle; 500 ml Bottle Top Filter (0.22 micron Cellulose
Acetate; Low Protein Binding Membrane; Sterilizing; Corning
Inc Cat# 430513).
Reagents:
~ Dulbecco's modified Eagle's medium nutrient mixture F-12
Ham; Sigma Cat# D-2906; stored at 2-8°C (equals a 1:1
mixture of Dulbecco's Modified Eagle's Medium (DME) and
Ham's F-12 Nutrient mixture in 15mM HEPES buffer without
phenol red and sodium bicarbonate).
~ Na2C03 (sodium bicarbonate); Sigma Cat # S-5761.
~ ITS+ Premix; BD Bioscience Cat # 354352; stored at 2-8°C;
stable for at least 3 months (the premix contains insulin,
transferrin, selenium, BSA and linoleic acid).
~ BD Nu-Serum; BD Bioscience Cat # 355100 (=100 ml_) or
355500 (=500 ml_); stored at -20°C; stable for at least 3 months;
upon arrival BD Nu-Serum should be aliquotted into 13 ml_
sterile storage vials under sterile conditions before freezing.
Final Concentrations of Components in Supplemented Medium:
~
15 mM HEPES
~
6.25 |jg/ml insulin
~
6.25 |jg/ml transferring
~
6.25 ng/ml selenium
~
1.25 mg/ml bovine serum albumin
~
5.35 |jg/ml linoleic acid
~
2.5 % Nu Serum
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Method, Procedures and Requirements.
Note: Due to possible varying hormone concentrations in different
batches of Nu-Serum, each new serum batch should be tested for
background hormone concentrations prior to use. It has been
shown that hormones present in medium are quickly metabolized
by the cells, and do not affect the response of the cells to
chemicals. However, for completeness reasons background
concentrations of testosterone and estradiol should be
documented. Furthermore, the same batch of Nu-Serum must be
used for each set of experiments.
Stock Medium (1L):
1. Place approximately 900 ml of ultra clean grade water (e.g.,
anopure) into the 1000 ml graduated cylinder.
2. Place the stir bar in the graduated cylinder and place the
graduated cylinder on the stir plate. Turn the stir plate on.
3. As the water stirs, add one bottle of DME/F12 powder (bottle
size for 1L).
4. Rinse the empty DME/F12 bottle with ultra clean grade water
to remove all traces of chemical and add this to the solution
once the initial powder added is totally dissolved.
5. Be sure to rinse the medium stuck on the sides of the
graduated cylinder down into the solution using ultra clean
grade water.
6. Add 1.2 g of sodium bicarbonate (Na2C03) and rinse the
weigh boat with water to remove all traces of chemical.
7. Adjust the pH of the solution to 0.1 to 0.3 below the desired
final pH of 7.4 with 1N HCI or 1N NaOH (whichever
appropriate).
8. Bring the final volume of the solution up to 1000 ml using
ultra clean grade water.
9. Filter the medium using a sterile 0.22 micron pore bottle top
filter into an amber autoclaved bottle. This should be done in
the biosafety cabinet because to maintain the sterility of this
solution.
10. Label the bottle as follows:
• DME/F12 Stock Medium
• H295R Cells
• "Filter Sterilized"
• pH = 7.4
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• Preparer's initials
• Date
• Expiration date
11. Medium should be stored at 4°C.
12. Stock medium can be used for up to 3 months, after that
new medium should be made.
Supplemented Medium (500 ml):
1. Thaw one vial (~13ml_) BD Nu-Serum using either a 37°C
water bath or incubator.
2. Allow the vial with 5 ml_ ITS+ premium mix to equilibrate to
room temperature - swirl gently to mix content.
3. Place an autoclaved 500 ml_ graduate cylinder in the
biosafety cabinet, and add 450 ml_ of stock medium.
4. Add 5 ml_ of the ITS+ premium mix and 12.5 ml_ of the BD
Nu-Serum to the cylinder containing the 450 ml_ of stock
medium, and bring the total volume up to 500 ml_.
5. Filter the medium in the biosafety cabinet into an autoclaved
500 ml amber bottle using a 0.22 micron bottle top filter.
6. Label the bottle as follows:
• DME/F12 Supplemented Medium
• H295R Cells
• "Filter Sterilized"
• pH = 7.4
• Preparer's initials
• Date
• Expiration date
7. Medium should be stored at 4°C.
8. Supplemented mediums can be used for up to 3 weeks, after
that new supplemented medium should be made.
Freeze Medium.
(i) Equipment, Materials and Reagents.
Equipment: Biosafety Cabinet.
Materials: 10 ml_ Strippettes; 15 ml_ Centrifuge Tubes (polypropylene;
sterile; plug seal cap; Corning Inc Cat# 430052); Autoclaved
100 ml_ Pyrex Bottle.
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Reagents: Sterile Stock Medium (see chapter 4.2); Dimethyl Sulfoxide
(DMSO) (A.C.S Reagent Baker Analyzed Cat# 9224-01); BD
Nu-Serum (see chapter 4.2).
(ii) Method, Procedures and Requirements.
1. All work is conducted under sterile conditions in a biosafety
cabinet.
2. The freeze media is made with H295R media supplemented
with 7.5% Nu-Serum and 5% Dimethyl Sulfoxide (DMSO).
3. Add 7.5 ml_ Nu-Serum and 5.26 ml_ DMSO to 92.5 ml_
H295R stock medium in a sterile 100 ml_ Pyrex bottle.
4. Mix well and aliquot freeze media into 15ml centrifuge tubes
(about 12 ml per tube).
5. Label the tubes as follows:
• Freeze Medium
• H295R Cells
• "Filter Sterilized"
• Preparer's Initials
• Expiration date
6. Store at -20°C until needed.
7. Freeze medium can be stored for up to 6 months.
Trypsin 1X.
(i) Equipment, Materials and Reagents.
Equipment: Biosafety Cabinet; Pipet-Aid.
Materials: 10 ml_ Strippettes; 15 ml_ Centrifuge Tubes (polypropylene;
sterile; plug seal cap; Corning Inc Cat# 430052); 500 ml Bottle
Top Filter (0.22 micron Cellulose Acetate; Low Protein Binding
Membrane; Sterilizing; Corning Inc Cat# 430513); 100 mL
Autoclaved Pyrex bottle.
Reagents: Trypsin-EDTA 10X (Life Technologies Inc. Cat # 15400-054);
Sterile PBS (see chapter 4.1).
(ii) Method, Procedures and Requirements.
1. All work is conducted under sterile conditions in a biosafety
cabinet.
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2. Filter 10X Trypsin-EDTA with a 0.22 micron filter and
transfer 10 mL of the filtered solution into 15 mL polystyrene
tubes.
3. Store unused filtered 10X trypsin at -20°C until needed.
4. Place 10ml of filtered 10X Trypsin-EDTA into a sterile 100
mL Pyrex bottle.
5. Bring the volume up to 100 mL using sterile PBS.
6. Aliquot the newly made 1X Trypsin into 15ml polypropylene
tubes.
7. Label the tubes with:
• Trypsin 1X
• Date
• Preparer's initials
• Expiration date
8. Store in the freezer at -20°C until needed.
9. Maximum storage time is 6 months.
Cell Maintenance and Culture Procedures.
(1) Purpose of H295R Cell Culture Protocol. The purpose of this protocol
is to define the appropriate culture of H295R cells obtained from ATCC
(CLR-2128) in preparation for the conduct of exposure experiments
utilizing the H295R Steroidogenesis Assay to identify chemicals with the
potential to interfere with estrogen and/or androgen production. This
protocol provides a consistent format for culturing H295R cell line. Such a
consistent format has been shown to be essential for the successful
conduct of the assay in terms of producing reproducible and accurate
results (Hecker et al. 2006 & 2008).
(2) Scope and Application of the H295R Cell Culture Protocol. The
protocol gives a detailed description of the methodology to culture and
maintain the H295R human adrenocortical carcinoma cell line (ATCC
CLR-2128). It provides a complete list of required reagents and solvents,
and describes the preparation of all solutions and reagents used in the
routine work with the cells. The protocol specifies cell maintenance
procedures including initiating cell cultures from frozen stock, the
cultivation and splitting of growing cells, and the freezing down of cells for
storage in liquid nitrogen. Finally, the protocol provides a description of
the proper maintenance of a cell culture diagram that allows for tracking
the progress of a cell line and provides a platform for recording all of the
work done with the cells.
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Stock medium is used as the base for the supplemented and freezing
mediums, which are used for a variety of different purposes in the cell
culture laboratory. Supplemented medium is a necessary component for
culturing cells. Freezing medium is specifically designed to allow for
impact-free freezing down of cells for long-term storage.
The procedure for starting the cells is to be used when a new batch of
cells is removed from liquid nitrogen storage for the purpose of culture and
experimentation. Splitting of the cells is necessary to ensure the health
and growth of the cells and to maintain cells for performing bioassays and
other testing. H295R cells will need to be frozen down to make sure that
there are always cells of the appropriate passage/age available for culture
and conduct of experiments. This procedure should also be implemented
when a cell line is not being actively used for research.
Cell Passage Nomenclature. In this protocol cell passages will be
labeled using a simplified version of the tracking system utilized in the Cell
Culture Diagram (Appendix 2) that is used in routine cell culture
procedures. A two-number system will be used that provides information
on the actual passage number as well as the passage number at which
the cells were frozen down. The first number indicates the actual cell
passage number and the second number describes the passage number
at which the cells were frozen down. The numbers are separated by a dot
{e.g., cells that underwent 4 passages after they were brought up into
culture again after being frozen down at passage 5 would be labeled
passage 4.5).
Starting Cells from the Frozen Stock.
(i) Equipment, Materials and Reagents.
Equipment: Biosafety Cabinet; Pipet-Aid; Centrifuge.
Materials: 10 ml Strippettes; Waste Container; 15 ml_ Centrifuge Tubes
(polypropylene; sterile; plug seal cap; Corning Inc Cat#
430052); 100 mm x 20 mm Cell Culture Dish (treated;
polystyrene, non-pyrogenic; sterile; Corning Inc Cat# 430167)
or Cell Culture Flask 75 cm2 (treated; polystyrene, non-
pyrogenic; sterile; Corning Inc Cat# 430641).
Reagents: Sterile Supplemented Medium (see chapter 4.2); H295R
cells (ATCC Cat # CRL-2128).
(ii) Method, Procedures and Requirements.
1. Aliquot 10 ml of the supplemented medium to a 15 ml
centrifuge tube. Use one centrifuge tube with medium for
each H295R vial that is being thawed.
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2. Remove a vial of H295R cells from liquid nitrogen storage.
3. Thaw the vial rapidly by agitation in a 37°C water bath or
using the warmth of hands. Remember to be very careful,
the vials are extremely cold and could cause damage to skin
if held in one position for too long. Thawing should be rapid
(within 40-60 seconds). As soon as the ice is melted,
remove the ampoule/vial from water bath and wipe it down
with 70% ethanol at room temperature and transfer to a
biosafety cabinet. All following steps must be done under
aseptic conditions.
4. In the biosafety cabinet, pipette the thawed cell solution into
the medium that was aliquoted in step 1.
Note: The thawed cell solution should be placed into the
aliquoted medium as quickly as possible. If the cells remain
in the freezing medium for too long the viability will be poor.
5. Centrifuge the cell suspension at 125 x g for 10 min, discard
the supernatant and resuspend the cells with 12 ml_ of
supplemented media by gentle swirling of the tube.
6. Transfer the cell suspension to a 100 mm x 20 mm culture
plate (further referred to as "plate").
Note: Culture flasks can also be used as an alternative to
culture plates. However, the amount of medium will need to
be adjusted if using a different size plate/flask with a different
surface area.
7. Label the plate with:
• Cell Type
• Date
• Initials of the person thawing the cells
• The plates will be given a plate designation after their
survival is assured.
8. Change the medium the next day (see following section on
"Maintaining and Sub-culturing the Cells" for the description
of H295R cell maintenance and medium change).
Maintaining and Sub-culturing the Cells.
(i) Equipment, Materials and Reagents.
Equipment: Biosafety Cabinet; Pipet-Aid, Incubator (37°C, 5% C02).
Materials: 10 ml_ Strippettes; 100mm x 20mm Cell Culture Dish (treated;
polystyrene, non-pyrogenic; sterile; Corning Inc Cat# 430167);
Waste Container; 15 and 50 ml_ Centrifuge Tubes
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(polypropylene; sterile; plug seal cap; Corning Inc Cat#
430052 [15 mL] and 430290 [50 mL]).
Reagents: Sterile Supplemented Medium (Subsection (e)(2)); Sterile 1x
Trypsin- EDTA (Subsection (e)(4)); Sterile PBS (Subsection
(e)(1)).
(ii) Method, Procedures and Requirements. H295R cells are
cultivated in an incubator at 37°C with 5% CO2 in air atmosphere.
Medium Renewal - Renew medium 2-3 times weekly:
1. Pre-warm supplemented medium to 37°C in a water bath or
incubator.
2. Wipe all tubes/containers off with 70% ethanol and transfer
cell culture plates and supplemented medium into a
biosafety cabinet.
3. Carefully pipette old medium off the culture plate without
disturbing cells.
4. Add 12 mL of fresh supplemented medium to the plate.
Splitting of Cells - Split the cells when they are close to confluence
(about 90% confluent):
1. Warm PBS and supplemented medium to 37°C in a water
bath or incubator.
2. Thaw and warm 1x Trypsin-EDTA at 37°C in a water bath or
incubator.
3. Wipe all tubes/containers off with 70% ethanol and transfer
cell culture plates and solutions into a biosafety cabinet.
4. Measure 15 ml of PBS for each plate to be split into a 50 ml
centrifuge tube (e.g., 45 mL for three plates; use 2nd tube for
4-6 plates, etc.).
5. Carefully pipette old medium off the culture plate without
disturbing cells.
6. Rinse each plate with 5 ml of sterile PBS, and discard PBS.
Rinse a total of 3 times. Make sure to change pipette tips
between each rinsing.
7. Add 1.5 mL of sterile 1x trypsin/plate and gently swirl plate to
distribute trypsin evenly (volume should be adjusted in
accordance with plate/flask size).
8. Wait for the cells to detach from the bottom of the plate.
Note: Plate can be placed in the incubator if the cells do not
separate easily.
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Note: Remember that the trypsin will kill the cells if left
on for too long. Watch the cells closely and stop the trypsin
action as soon as the cells have separated from the
plate/flask (typically this should not take longer than 4-5
minutes). Cells should not been exposed to trypsin for
more than 10 minutes!
9. Stop the trypsin action with 10.5 mL of supplemented
medium (once again the volume will need to be adjusted for
a different sized plate/flask).
10. Place the appropriate amount of cell solution in the new
plate/flask. The amount of cell solution should be adjusted
so that the cells are confluent within 5-7 days. The
recommended sub-cultivation ratio is 1:3 to 1:4.
11. Label the plate with:
• Cell Type
• Date
• Initials of the person splitting the cells
• Unique identifier code containing ATCC Lot number,
Freeze Down ID, Passage #, Total # of Passages since
original ATCC cell batch was started in cell culture,
Plate ID (Subsection (f)(6) and Appendix 1: Cell Culture
Diagram)
• Plate designation
Freezing H295R Cells (Preparing Cells for Liquid Nitrogen Storage).
(i) Equipment, Materials and Reagents.
Equipment: Biosafety Cabinet; Pipet-Aid; Controlled Rate Freezing
Container (1 Degree C Freezing Container; Nalgene Cat#
5100-0001); Centrifuge, Liquid Nitrogen Tank.
Materials: 10 ml Strippettes, Sterile Cryrogenic Vials (Polypropylene,
Biohit Inc. Cat# 4503-1); Waste Container, 15 mL Centrifuge
Tubes (polypropylene; sterile; plug seal cap; Corning Inc Cat#
430052).
Reagents: Supplemented Medium (Subsection (e)(2)); Sterile IxTrypsin-
EDTA (Subsection (e)(4)); Freeze Medium (Subsection (e)(3));
Sterile PBS (Subsection(e)(1)).
(ii) Method, Procedures and Requirements.
Page 14
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1. Follow the procedure for splitting cell lines (Subsection (f)(4))
through step nine in the method (stopping of trypsin action
with 10.5 mL supplemented medium).
2. Pipette all of the cell solution into a sterile 15 ml centrifuge
tube that is labeled with the plate identifier code.
3. Centrifuge tube with cells for 5 minutes at 350 x g at room
temperature.
4. Upon removal from the centrifuge there should be a pellet of
cells in the bottom of the centrifuge tube. If not, spin again
under the same conditions.
5. Pipette off the supernatant and place it into the waste
container in the biosafety cabinet.
Note: Be sure not to suck up the pellet. If this does happen,
re-suspend the pellet in the medium and spin down in the
centrifuge again.
6. Re-suspend the pellet of cells in 1 ml of the appropriate cell
freezing medium.
7. Transfer the solution to a sterile cryogenic vial and label
with:
• Cell Type.
• Date.
• Initials of the person freezing the cell line down.
• Unique identifier code containing ATCC Lot number,
Freeze Down ID, Passage #, Total # of Passages since
original ATCC cell batch was started in cell culture,
Plate ID (Subsection (f)(6) and Appendix 1: Cell Culture
Diagram).
8. Place the vial(s) into the controlled rate freezing container.
9. Put the container into a -80°C freezer for 24 hours.
10. After 24 hours in the freezer, transfer to liquid nitrogen for
storage.
11. The storage in liquid nitrogen vapor phase is recommended
instead of having the cryogenic vials submerged in the liquid
nitrogen fluid.
Cell Culture Diagram. Cell culture diagrams are useful and necessary
for the smooth operation of the cell culture laboratory. They are used for a
variety of reasons including: (1) They allow for tracking the progress of a
cell line and notice problems or incongruencies, which may arise over time
(These are very important to note), and (2) They also provide a platform
for recording all of the work done with the cells.
Page 15
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1. It is recommended that the following information from the
saved cryovial is tracked:
• Cell Type
• Date the cells were frozen
• ATCC Lot number
• Freeze Down ID / Passage # / Total # of Passages
since original ATCC cell batch was started in cell
culture / Plate ID
2. Each time the cells are split the cell passage number and
total number of passages are increased by one. Each time
the cells are frozen the freeze down ID (Greek letter) is
increased by one starting with A and continuing with
B, r, A, E etc. (see Appendix 1 for key). When the cells that
have been frozen are started in culture, the passage number
is reset to 1, and the total number of passages is continued.
Each different plate/flask is labeled with another letter with
the first being A, the second B, and so on.
3. Each data sheet in the study will contain the information on
the cells used {i.e., Cell Type, Date the cells were frozen,
ATCC Lot number, Freeze Down ID, Passage #, Total # of
Passages since original ATCC cell batch was started in cell
culture, and Plate ID). As the cells continue to grow, a
record is made of each operation such as changing the
media, freezing cells, splitting the cells etc. (See Appendix 2
for a sample diagram).
4. Continue with the cell culture diagram until the cells are no
longer being used or are frozen.
(7) Records, Documentation and QC Requirements. When preparing
media, remove label from the bottle or packet and place in notebook, with
your initials and date that the medium was made. When starting new
cells, start a "cell culture diagram" for the cell line (see the SOP for starting
and maintaining a "cell culture diagram"). Record the culturing, splitting
and freezing of the cells on the "cell culture diagram". Be sure to include
the date that the medium was changed, cells were split or frozen. It is
recommended that the liquid nitrogen Dewar contents log book is updated
if removing a vial to start new batch of cells or when transferring cryogenic
vials with frozen cells to the liquid nitrogen. Any anomalies and/or
deviation from the specified are documented.
Pre-test Procedures.
(1) Solutions and Reagents for Testing.
Page 16
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(i) Equipment, Materials and Reagents.
Equipment: Pipet-Aid, Stir Plate; Analytical Balance.
Materials: Weigh Boats; Chemical Spatula; Pasteur pipettes; Waste
Container, 2,10, 200 and 1,000 ul Pipettes, Sterile Pipette
Tips, Sterile Amber Glass Vials (National Scientific Company;
Cat# C4000-2W).
Reagents & Chemicals:
Dulbecco's Phosphate Buffered Saline (PBS)*
Stock Medium*
Supplemented Medium*
Sterile 1x Trypsin*
DMSO (Aldrich, Cat. No. D2438)
Forskolin (MW= 410.50); 4.1050 mg
Prochloraz (MW= 376.67); 3.7667 mg
Test Chemicals
* The preparation and storage conditions for Dulbecco's Phosphate Buffered
Saline (PBS), Stock and Supplemented Medium, and trypsin stock are
described in detail in the in Section (e).
(ii) Method, Procedures and Requirements.
Step 1: Prepare 100 mM stock concentrations of forskolin, prochloraz and
test chemicals dosing solutions in DMSO. Weigh out the
appropriate amount of forskolin (4.1050 mg), prochloraz (3.7667
mg) and test chemicals (MWx 0.00001) in tared vials. Add 100 |jL
of DMSO to the forskolin, prochloraz and all test chemical vials, cap
and vortex to dissolve each substance in the DMSO. This results in
100 mM stock solutions for forskolin and prochloraz. For all test
chemicals this results in the Stock 1 Test Solution.
Step 2: Dilute these stock solutions as follows:
~ Forskolin: Dilute 100 mM stock solution 1:10 (10 |jL of 100
mM Stock 1 + 90 |jL DMSO) to make 100 |jL of a 10 mM
solution. Dilute 10 |jL of this 10 mM solution 1:10 to make 100
uL of a 1 mM solution.
~ Prochloraz: Dilute 100 mM stock solution 1:10 (10 |jL of 100
mM Stock 1 + 90 |jL DMSO) to make 100 |jL of a 10 mM
solution. Dilute 10 |jL of this 10 mM solution 1:10 to make a 1
mM solution. Dilute 10 |jL of this 1 mM solution 1:10 to make
a 0.1 mM solution.
~ Test Chemicals: Dilute Stock 1 1:10 (10 |jL of 100 mM Stock
1 + 90 uL DMSO) to make 100 |jL of Stock 2 solution.
Page 17
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Continue diluting these stock solutions in similar manner with
DMSO until a total of seven (7) dilutions have been made
(Stock 1 - Stock 7). Dilutions should be made serially from
the next greater concentration (e.g., a Stock 4 would be made
by adding 10 |jL of a Stock 3 to 90 |jL of DMSO).
Step 3: Label each vial as follows:
~ Forskolin & prochloraz: Chemical name, date the solution
was made, concentration of chemical in mM, type of solvent
(DMSO) and preparers initials.
~ Test chemicals: Chemical ID number, date the solution was
made, Stock identifier (e.g., Stock 1), type of solvent
(DMSO) and initial.
Step 4: Store the stock solutions at 4°C.
Requirements Before Initiating Testing.
~ Prior to the initiation of cell culture and any subsequent testing, each
laboratory is expected to demonstrate the sensitivity of its hormone
measurement system (Subsection (g)(2)(i)).
~ If antibody-based hormone measurement assays are to be used, it is
recommended that the chemicals to be tested be analyzed for their
potential to interfere with the measurement system used to quantify
testosterone and estradiol, as outlined in Subsection (g)(2)(ii) prior to
initiating testing.
~ Prior to conducting testing for the first time, the laboratory is expected
to conduct a qualifying experiment demonstrating that the laboratory
is capable of maintaining and achieving appropriate cell culture and
experimental conditions required for chemical testing, as described in
Subsection (g)(2)(iii).
~ In addition to being run in parallel with each test run, it is
recommended that a QC control plate be run before using a new
batch of cells to evaluate the performance of the cells, as described
in Subsection (g)(2)(iv).
(i) Performance of Hormone Measurement System. Each
laboratory may use a hormone measurement system of its choice
for the analysis of the production of T and E2 by H295R cells. Prior
to the initiation of cell culture and any subsequent test runs, it is
expected that each laboratory demonstrate the conformance of
their hormone measurement system (e.g., ELISA, RIA, LC-MS) with
the QC criteria defined in Table 1 by analyzing supplemented
medium spiked with an internal hormone control.
Page 18
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Due to the cross-reactivity of some of the antibody based hormone
ELISA's and RIA's with hormone metabolites/conjugates produced
by the H295R cells, an extraction of the medium is required prior to
the measurement of hormones if an assay is used that employs
antibodies.
Table 1. Performance Criteria for Hormone Measurement Systems.
Parameter
Criterion
Method Detection Limit
Testosterone: 100 pg/mL; Estradiol: 10 pg/mL a
Spike Sample Recovery
Supplemented medium are spiked with at least two
concentrations of each hormone of interest (T: 500 and 2500
pg/mL; E2: 50 and 250 pg/mL). When analyzed with the
hormone measurement assay, the average recovery rates (based
on triplicate measures) for the spiked amounts of hormone are
expected not to deviate more than 30% from nominal
concentrations.
Hormone Cross reactivity (only
antibody based systems)
No significant (>30% of basal hormone production of the
respective hormone) cross-reactivity with any of the hormones
produced by the cells are expected to occur. These include:
cholesterol, pregnenolone, progesterone, 11-
deoxycorticosterone, corticosterone, aldosterone, 17alpha-
pregnenolone,17alpha-progesterone, deoxycortisol, Cortisol,
DHEA, androstenedione, estrone13
a Note: Method detection limits shown here are based on the basal hormone production values provided
in Table 4, and are performance based. If greater basal hormone production can be achieved, a higher
MDL is acceptable provided the criteria in Table 4 are met.
faSome T and E2 antibodies may cross-react with androstendione and estrone, respectively, at a greater
percentage In such cases it is not possible to accurately determine effects on 17p-HSD. However, the
data can still provide useful information regarding the effects on estrogen or androgen production in
general. In such cases data should be expressed as androgen/estrogen responses rather than E2 and T.
One additional criterion for the acceptance of data is the validity of
the SC in terms of its position within the standard curve range. The
SC is expected to be within the 75% range below the upper part
(maximum optical density [OD] or similar response measured by
hormone measurement system) and 75% above the lower part
(minimum OD or similar response measured by hormone
measurement system) of the linear range of standard curve for
inducers, and inhibitors, respectively (Figure 1). Dilutions of
medium (extracts) in the hormone measurement assay are to be
selected accordingly.
Page 19
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Acceptable Linear Range Acceptable
Range for SC -
Inducers ^
Range for SC -
" Inhibitors
Min
Max
0.4
Center
L35
0.3
L25
0.2
5
1
L05
100% Ram
0
0
1
2
3
4
log Concentration
Figure 1. Example of hormone measurement system standard curve indicating
expected range of hormone concentration of solvent control (SC) sample (not corrected
for dilution in assay). Max = upper limit of linear range; Min = lower limit of linear range.
Grey shaded area = 75% range below the maximum OD of the linear part of the
standard curve. Diagonally striped area = 75% range above the minimum OD of the
linear part of the standard curve.
(ii) Chemical Hormone-Assay Interference Test. If antibody-based
hormone measurement assays are to be used, prior to initiation of
testing, it is recommended that each chemical be tested for
potential interference with the hormone measurement system being
utilized. It has been previously shown that that some chemicals
can interfere with antibody-based assays such as ELISAs and RIAs
(Shapiro and Page 1976). This "chemical interference test" will be
conducted as described for the analysis of medium samples
(Subsection (h)(5)). Prior to extraction and analysis, chemical
spiked medium is prepared as follows.
(A) Equipment, Materials, and Reagents.
Equipment: Biosafety Cabinet; Pipet-Aid, Vortex.
Materials: 10 ml_ or 25 ml_ Strippettes; Waste Container; 15 or
50 ml_ Centrifuge Tubes (polypropylene; sterile; plug
seal cap; Corning Inc Cat# 430052 [15 ml_] or 430290
Page 20
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[50 mL]); 2 |liL and 1000 |liL Pipettes; Sterile Pipette
Tips, Eppendorf Tubes (1.5 mL).
Reagents: Sterile Supplemented Medium (Subsection(e)(2);
DMSO (Aldrich, Cat. No. D2438); Test chemical stock
solutions (serial dilutions 1 -6; see Subsection (g)(1));
Testosterone; 17(3-Estradiol; Ethanol (100%).
(B) Method, Procedures and Requirements.
Step 1: Prepare Stock Solutions:
~ Dissolve 1 mg of T and E2 each in 1 mL of 200 proof
ethanol in Eppendorf tubes {Primary Hormone
Stocks).
~ Dilute 10 |jI of Primary Hormone Stocks from previous
step each in 990 |jl of a supplemented medium ethanol
solution (75 % EIA / 25 % ethanol) and vortex
(¦Secondary Hormone Stocks)
~ Dilute 10 |jI of Secondary Hormone Stocks from
previous step each in 990 nl supplemented medium
and vortex {Tertiary Hormone Stocks).
~ Dilute 100 |jl of Tertiary Hormone Stocks from
previous step each in 900 nl supplemented medium
and vortex, resulting in a hormone stock concentration
of 10 ng / mL {Final Hormone Stock Solution).
Step 2: Prepare two Eppendorf Tubes per test chemical for initial
interference test.
Step 3: Add 999 |jL of supplemented medium to tube 1 and 779 |jL
of supplemented medium to tube 2.
Step 4: Add 200 |jL of testosterone Final Hormone Stock Solution
and 20 |jL of estradiol Final Hormone Stock Solution to tube
2.
Step 5: Add 1 |jL of Test Chemical Stock Solution 1 (greatest
concentration) to tubes 1 and 2.
Step 6: Vortex thoroughly for >15 seconds.
Step 7: Extract medium supplemented with Test Chemical and
Hormone Stock Solutions produced in previous steps and
analyze for hormones as described in Subsection (h)(5).
Page 21
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Note: If significant interference of chemical occurs as determined
by hormone analysis (significant interference is defined as > 30% of
basal hormone production for testosterone and/or estradiol), it is
recommended that the Chemical Hormone Assay Interference Test
be repeated for all Test Chemical Stock Solution dilutions to identify
the threshold dose at which significant interference occurs. If
significant interference of a test chemical with a hormone
measurement system occurs at more than one non-cytotoxic
concentration, it is recommended that a different hormone
measurement system be used.
(iii) Laboratory Proficiency Test. Before testing unknown
substances, a laboratory is expected to demonstrate that it is
capable of achieving and maintaining appropriate cell culture and
test conditions required for the successful conduct of the assay. As
the performance of an assay is directly linked to the laboratory
personnel conducting the assay, it is recommended that these
procedures be repeated if a change in laboratory personnel occurs.
This qualification test will be conducted under the same conditions
listed in Section (h) by exposing cells to six increasing
concentrations of the model inducer forskolin and the model
inhibitor prochloraz. These qualification tests will be done under a
somewhat different exposure regime and plate layout compared to
the standard test chemical exposure procedure in that the
concentrations are more closely distributed about the expected
values of the EC50 of each of the control chemicals than would be
the case for an initial run with an unknown chemical.
Table 2. Dosing Schematic for
the Laboratory Proficiency SI
udy.
1
2
3
4
5
6
A
DMSO
DMSO
DMSO
For 10 or
For 10 or
For 10 or
M
1 (jL
1 (jL
1 (jL
Pro 3
Pro 3
Pro 3
B
For 0.03 or
For 0.03 or
For 0.03 or
For 1 or
For 1 or
For 1 or
Pro 0.01
Pro 0.01
Pro 0.01
Pro 0.3
Pro 0.3
Pro 0.3
For 0.3 or
For 0.3 or
For 0.3 or
For 0.1 or
For 0.1 or
For 0.1 or
Pro 0.1
Pro 0.1
Pro 0.1
Pro 0.03
Pro 0.03
Pro 0.03
D
For 3 or
Pro 1
For 3 or
Pro 1
For 3 or
Pro 1
Blank
Blank
Blank
Exposure of H295R to either forskolin (For) or prochloraz (Pro) in a
24 well plate during the laboratory proficiency test. Dosing is in |jM
for all test chemical doses. Note: Doses will be administered in
DMSO at 0.1 % v/v per well. The DMSO solvent control will receive
1 |jL of DMSO only. Nothing is added to the Blank wells. Separate
plates are to be run for forskolin and prochloraz.
Page 22
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The effective concentrations expressed as 50% of the maximum
response (EC50S) are calculated using standard procedures (e.g.,
Probit analysis or moving average method) and compared with the
values in Table 3. The data are expected to fall within the following
ranges:
Table 3. EC50 Ranges for Control Substances.
EC50 (MM)
T
E2
Prochloraz
0.01 -0.1
0.03-0.3
Forskolin
0.2-2.0
O
CO
I
CO
0
(iv) Quality Control Plate. There are two uses of the quality control
(QC) plate:
(A) It is recommended that H295R cell performance be assessed
for possible changes in hormone production as a function of
cell age prior to using a new ATCC batch or after using a
previously frozen stock of cells for the first time, unless the
laboratory proficiency test (Subsection (g)(2)(iii)) has been run
with that batch of cells. To verify that the performance of
H295R Cells under Standard Culture Conditions is meeting
the QC requirements, a subset of passage five (5.0) cells is
run in a QC plate. Laboratories that have already frozen
passage five (5.0) cells can thaw one of the frozen batches,
grow it for three (3) passages (passage # 3.5), and use these
cells for the QC run.
(B) A QC plate provides the positive controls for the assay when
testing chemicals and is included as part of each test run.
The QC plate is a 24 well plate shown in Table 4 and is incubated,
dosed, and assessed (cell viability/cytotoxicity, hormones extraction
and hormone analysis protocols) in the same manner as test plates
described in Section (h). The QC plate will be dosed with a known
inducer (forskolin) and inhibitor (prochloraz) of E2 and T synthesis
each at two different doses of these compounds.
Page 23
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Table 4. Quality Control Plate Layout for Testing Performance of Unexposed H295R
Cells and Cells Exposed to Known Inhibitors (PRO = prochloraz) and Stimulators (FOR
= forskolin) of E2 and T Production.
1
2
3
4
5
6
A
Blank3
Blank3
Blank3
Blank3
+ MeOHb
Blank3
+ MeOHb
Blank3
+ MeOHb
B
DMSO
1 |JL
DMSO
1 |jL
DMSO
1 |jL
DMSOIjjL
+ MeOHb
DMSOIjjL
+ MeOHb
DMSO 1 [it
+ MeOHb
C
FOR 1 |JM
FOR 1 |jM
FOR 1 |jM
PRO 0.1 |jM
PRO 0.1 |jM
PRO 0.1 |JM
D
FOR 10 |JM
FOR 10 |jM
FOR 10 |jM
PRO 1 |jM
PRO 1 |jM
PRO 1 |JM
aBlank wells receive medium only.
bA 70% methanol (MeOH) solution will be added to all MeOH wells after termination of the exposure
experiment and removal of medium.
Criteria for the QC plate are provided in Table 5.
Table 5. Criteria to be Met on Eac
i QC Plate.
Testosterone
Estradiol
Minimum Basal Production
500 pg/mL
40 pg/mL
Basal Production
> 5-times MDL
> 2.5-times MDL
Induction (10uM forskolin)
> 2-times SC
> 7.5-times SC
Inhibition (1uM prochloraz)
< 0.5-times SC
< 0.5-times SC
The minimum basal hormone production must be met in both
solvent control and blank wells.
Note: In some cases basal E2 production may be still less than
desired (<40 pg/mL) regardless of cell density and age/generation.
In such cases 22-R Hydroxycholesterol can be added to the
supplemented medium at concentrations between 20 and 40 /llM to
increase basal production of E2. In any case, the performance of
the assay must be demonstrated in the proficiency test (Subsection
(g)(2(iii)) prior to the conduct of any experiment.
(h) Test Procedures.
(1) Plating and Pre-lncubation of Cells.
Note: Due to changes in estradiol producing capacities of the cells
with age (i.e., cell passage) (Heckeret al. 2006), cells are cultured
following a specific protocol before they are used for the conduct of
experiments: After initiation of an H295R culture from an original
ATCC batch following the procedures outlined in the H295R Culture
Protocol, cells are grown for five (5) passages (cells need to be split
4-times). These cells are frozen in liquid nitrogen (Subsection (f)(5)
Page 24
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Freezing H295R Cells). Cells starting from these frozen batches,
following the procedures described Subsection (f)(3) Starting Cells
from Frozen Stock, need to be cultured for at least four (4)
additional passages (passage # 4.5) before they can be used to
conduct the assay. The maximum passage used in testing is 10
(passage # 5.10). Before using cells in a chemical exposure
experiment, it is recommended that a QC plate be run to verify
"readiness" of the cells for conducting the assay.
Equipment, Materials and Reagents.
Equipment: Biosafety Cabinet; Pipet-Aid; Centrifuge; Microscope.
Materials: 10 mL Strippettes; Waste Container; 15 and 50 mL
Centrifuge Tubes (polypropylene; sterile; plug seal
cap; Corning Inc Cat# 430052 [15 mL] and 430290
[50 mL]); 24-well Tissue Culture Plates (Corning Inc.
Costar Cat# 524); Sterile 1.5 mL Eppendorf tubes;
10|aL, 100 |liL, 1mL Pipettes; Sterile Pipette Tips;
Hemocytometer.
Reagents: Sterile Supplemented Medium (Subsection (e)(2);
Passage 4.5 to 10.5. NCI-H295R cells (ATCC Cat #
CRL-2128) cultured under standard conditions as
described in the H295R culture protocol; Sterile 1x
Trypsin- EDTA (Subsection (e)(4)); Sterile PBS
(Subsection (e)(1)).
Methods, Procedures and Requirements.
Step 1: Pre-warm PBS and supplemented medium to 37°C in a
water bath or incubator.
Step 2: Thaw and pre-warm 1x Trypsin-EDTA at 37°C in a water
bath or incubator.
Step 3: Wipe all tubes/containers off with 70% ethanol and transfer
cells and solutions into a biosafety cabinet.
Step 4: Remove a H295R cell culture plate cultured under standard
conditions as outlined in the H295R culture protocol from
incubator and place in biosafety cabinet.
Note: The number of cell culture dishes that will be needed
for an experiment depends on the number of plates needed
for the exposure experiment and the confluency of the cells
in the culture dishes. General rule: use 1 cell culture dish
Page 25
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(100 mm) of 95-100% confluent cells to plate two 24-well
plates at a target density of 200,000 to 300,000 cells per mL.
Step 5: Prepare centrifuge tube or small sterile glass bottle with
about 11 mL media for every plate to be trypsinized.
Step 6: Measure 15 mL of PBS for each plate to be split to a 50 mL
centrifuge tube (e.g., 45 mL for three plates; use 2nd tube for
4-6 plates, etc.).
Step 7: Carefully pipette old medium off the culture plate without
disturbing cells.
Step 8: Rinse plate with 5 mL of sterile PBS, and discard PBS.
Rinse a total of 3 times. Make sure to change pipette tips
between each rinsing.
Note: Rinse gently to avoid detaching cells from the plate
by adding PBS down the side of the well.
Step 9: Add 1.5 mL of sterile 1x trypsin/plate and gently swirl plate to
distribute trypsin evenly (volume should be adjusted in
accordance with plate/flask size).
Step 10: Wait for the cells to detach from the bottom of the plate.
Note: Plate can be placed in the incubator if the cells do not
separate from the plate easily. Also, need to be careful not
to knock or shake plate to avoid clumping of cells.
Note: Remember that the trypsin will kill the cells if left on
for too long. Watch the cells closely and stop the trypsin
action as soon as the cells have separated from the
plate/flask (typically this should take not longer than 4-5
minutes). The maximum exposure time to trypsin that
cells can tolerate is 10 minutes!
Step 11: Harvest the trypsinized cells and transfer them into the
centrifuge tube or bottle with medium.
Step 12: Thoroughly mix (aspirate using 10 mL stripette) the cell
suspension of medium and trypsinized cells to make the
content homogenous.
Note This is important for accurate cell counts because the
cells tend to clump.
Step 13: Take a small sub-sample (30-50 |jL) of the well-mixed cell
suspension and transfer to an Eppendorf tube.
Page 26
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Step 14: Clean the hemocytometer and the cover glass with 70%
Ethanol. Add 10 jjl of the cell solution from the Eppendorf
tube under the cover glass in the hemocytometer. Count the
cells - at least 3 squares from each side of the
hemocytometer. Calculate the mean of all the counts. The
cell density is the mean cell count x 1Q4. For example if the
mean count is 110, there are 1,100,000 cells/mL = 1.1 x106
cells/mL
Step 15: Calculate the volume of cell solution needed for the selected
number of 24-well plates (calculate 1.2 mL media/well).
Step 16: Cells need to be seeded at 200,000 to 300,000 cells/mL
medium resulting in approximately 50-60% confluency in the
wells at 24 hours (Figure 2).
Note: 50 - 60% confluency in the wells at 24 hours is the
preferred density of cells for optimal hormone production in
the medium. At higher densities cells tend to be affected by
hormonal feedback or other mechanisms and T as well as
E2 production patterns are altered. Before conducting the
assay the first time, different seeding densities between
200, 000 and 300,000 cells per mL should be tested, and the
density resulting in 50 to 60% confluency in the well at 24
hours should be selected for further experiments.
A
Figure 2. Photomicrograph of H295R Cells at a Seeding Density of 50 - 60% in a 24
Well Culture Plate at 24 hours.
Page 27
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Step 17: Dilute the cell solution to the desired plating density.
Thoroughly mix the cell solution to assure homogenous cell
density.
Step 18: Plate the cells with 1 ml_ of the cell solution/well.
Step 19: The new plates should be labeled with the cell type,
preparer's initials and plating date. Individual wells
(samples) should be labeled with sample name.
Note: All of the above steps with the exception of steps 14
& 15 (counting of cells) need to be conducted under sterile
conditions in a biosafety cabinet.
Step 20: Incubate seeded plate in incubator at 37°C under a 5% CO2
in air atmosphere for 24 hours.
Exposure of Cells.
(i) Equipment, Materials and Reagents.
Equipment: Biosafety Cabinet; Pipet-Aid, Incubator (37°C, 5%
CO2); Vortex, Microscope.
10 ml_ and 25 ml_ Strippettes; Waste Container; 15 and
50 ml_ Centrifuge Tubes (polypropylene; sterile; plug
seal cap; Corning Inc Cat# 430052 [15 ml_] and 430290
[50 ml_]); 24-well Tissue Culture Plates (Corning Inc.
Costar Cat# 524) seeded at 200,000 to 300,000 cells
per well with H295R cells and pre-incubated for 24
hours; 2 iliL and 10|uL Pipettes; Sterile Pipette Tips.
Sterile Supplemented Medium (Subsection (e)(2));
DMSO; Test chemical stock solutions (serial dilutions 1
-6; Subsection (g)(1)).
Materials:
Reagents:
(ii) Methods, Procedures and Requirements.
Step 1: Remove cells from incubator that have been pre-incubated
for 24 h and check under microscope to assure good
condition (attachment, morphology) prior to dosing.
Step 2: Place cells in biosafety cabinet and remove old medium.
Step 3: Add new medium (1 mL/well) to all wells.
Note: Alternatively, a mastermix containing 3.94 mL of
medium plus 4 pL of the respective chemical stock solution
Page 28
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in DMSO can be prepared prior to dosing the cells. Then, 1
mL of the appropriate mastermix is to be dispensed per
replicate well of each dose. If this approach is chosen, omit
step 4.
Step 4: Dose cells by adding 1 |jl of the appropriate stock solution in
DMSO per 1 mL medium (well volume). This results in a
final concentration of DMSO of 0.1%.
Step 5: For solvent controls, add 1 pi DMSO per 1 mL medium (well
volume) directly into the well.
Step 6: Dose cells according to the exposure layout as indicated in
Table 6.
Table 6. Dosing Schematic for the Exposure of H295R to Test Chemicals in a 24 Well
Plate.
1
2
3
4
5
6
A
DMSO
DMSO
DMSO
Stock 4
Stock 4
Stock 4
M
1 Mi-
1 |JL
1 Mi-
1 Mi-
1 Mi-
1 Mi-
p
stook 1
Stockl
Stock 1
Stock 5
Stock 5
Stock 5
D
1 Mi-
1 Mi-
1 Mi-
1 Mi-
1 Mi-
1 Mi-
p
stook 2
stook 2
Stock 2
Stock 6
Stock 6
Stock 6
U
1 Mi-
1 Mi-
1 Mi-
1 Mi-
1 Mi-
1 Mi-
n
stook 3
Stock 3
Stock 3
Stock 7
Stock 7
Stock 7
u
1 |jL
1 ml
1 ml
1 ml
1 ml
1 ml
Dosing is calculated based on a total volume of 1 mL per well.
Stock 1-7: 1 |jl_ of appropriate stock solution needs to be added to each well.
Step 7: Incubate dosed plate in incubator at 37°C under a 5% CO2 in
air atmosphere for 48 hours.
(3) Exposure Termination and Medium Storage.
(i) Equipment, Materials and Reagents.
Equipment: Microscope; -80°C freezer.
Materials: 1.5 mL Eppendorf tubes; 1 mL Pipette, Pipette Tips; 24-
well Tissue Culture Plates (Corning Inc. Costar Cat#
524) seeded at 200,000 to 300,000 cells per well with
H295R cells and exposed for 48 hours.
(ii) Method, Procedures and Requirements.
Step 1: Remove exposure plate from incubator and check every well
under the microscope for cell condition (attachment,
morphology, degree of confluence), look for signs of
cytotoxicity. Record your observations.
Page 29
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Step 2:
Label Eppendorf tubes appropriately.
Step 3: Split medium from each well into two equal amounts
(approx. 490 |jL each) and transfer to two separate
Eppendorf tubes appropriately labeled (e.g., a & b).
Step 4: Freeze media at -80°C until further processing (Subsection
(h)(5)).
Step 5: Immediately after removing medium conduct a cell viability
test with each exposure plate (Subsection (h)(4)).
IMPORTANT: Make sure that cells do not dry out, remove
medium a row or column at a time and add 200 pL PBS with
Ca+ and Mg+ to each well.
Cell Viability Measurements. A cell viability/cytotoxicity assay of choice
can be used to determine to the potential impact of the test chemical on
cell viability. The assay must provide a true measure of the percentage of
viable cells present in a well, or it must be shown to be directly
comparable to (a linear function of) the Live/Dead® Assay described
below. An alternative assay that has been shown to work equally well as
the Live/Dead® Assay is the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide] test (Mosman et al. 1983).
Note: The assessment of cell viability using the above methods is a
relative measurement that does not necessarily exhibit linear relationships
with the absolute number of cells in a well. Therefore, a subjective parallel
visual assessment of each well by the analyst should be conducted, and
digital pictures of the SCs and the two greatest non-cytotoxic
concentrations are to be taken and archived to enable later assessment of
true cell density if this should be required.
(i) Live/Dead® Cell Viability/Cytotoxicity Assay. The LIVE/DEAD
Viability/Cytotoxicity Kit (Molecular probes, Eugene OR, USA, Cat#
L-3224) gives a simultaneous determination of live and dead cells
with two probes that measure two recognized parameters of cell
viability: intracellular esterase activity and plasma membrane
integrity. Live cells are distinguished by the presence of ubiquitous
intracellular esterase activity as determined by the enzymatic
conversion of the virtually non-fluorescent cell-permeate calcein AM
to the intensely fluorescent calcein. The polyanionic dye calcein is
well retained within live cells, producing an intense uniform green
fluorescence in live cells (Ex/Em ~495 nm/~515 nm). Ethidium
homodimer 1 (EthD-1) enters cells with damaged membranes and
undergoes a 40-fold enhancement of fluorescence upon binding to
nucleic acids, thereby producing a bright red fluorescence in dead
cells (Ex/Em ~495 nm/~635 nm). EthD-1 is excluded by the intact
Page 30
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plasma membrane of live cells. The determination of cell viability
depends on these physical and biochemical properties of cells.
Cytotoxic events that do not affect these cell properties may not be
accurately assessed using this method. Background fluorescence
levels are inherently low with this assay technique because the
dyes are virtually non-fluorescent before interacting with cells.
The purpose of this section is to provide a consistent format for
cytotoxicity testing with H295R cell line using a LIVE/DEAD
Viability/Cytotoxicity Kit. The cytotoxicity testing will be conducted
in the chemical exposure plate and must be conducted immediately
after termination of the exposure experiments.
The methods described here are have been optimized based on
the use of 24 well plates and the Fluoroskan Ascent plate reader.
(A) Equipment, Materials and Reagents.
Equipment: Vortex; Microscope; Eppendorf Multipipette; Plate
reading fluorometer (Note: this protocol has been
tested with the Fluoroskan Ascent Fluorometric
Microtiter Plate Reader (Thermo Electron
Corporation)).
Materials: 100-1000, 20-200, 2-20 and 0.5-2 |liI Pipettes, Pipette
Tips; 15 and 50 ml_ Centrifuge Tubes (polypropylene;
sterile; plug seal cap; Corning Inc Cat# 430052 [15
ml_] and 430290 [50 ml_]); 24-well Tissue Culture
Plates (Corning Inc. Costar Cat# 524) seeded at
200,000 to 300,000 cells per well with H295R cells
and exposed for 48 hours.
Reagents: Cell viability assay reagents; sold either as a kit from
Molecular Probes (#L-3224; Eugene, OR) or as
individual components:
~ Calcein AM (Molecular Probes #C-3100);
MW = 994.87; made up as 4000x (2 mM) stock
(50 Mg/12.56 mL DMSO).
~ Ethidium homodimer I (Molecular Probes #E
1169); MW = 857; made upas 2000x (1 mM)
stock in DMSO.
Dulbecco's phosphate-buffered saline with Ca2+ and
Mg2+ (PBS; Subsection (e)(1)).
(B) Method, Procedures and Requirements.
Preparation Steps (prior to assay):
Page 31
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Step 1: Inspect plates visually with and without microscope - check
degree of confluence, homogeneity from well-to-well, and
any signs of cytotoxicity or altered morphology. Note all
observations in laboratory notebook.
Step 2: Prepare viability assay reagent (refer to Supplies and
Biochemicals Section): Each plate will need 16 ml_ (24 wells
* 600 |jL) plus a little extra (1.6 ml_).
Step 3: Dilute the appropriate amounts of calcein and Ethidium with
the appropriate volume of of PBS with Ca2+ and Mg2+:
- Calcein stock (|jL): 0.5 |jL/mL
- Ethidium bromide stock (|jL) 2.0 |jL/ml_
CAUTION: Ethidium bromide homodimer is a powerful
mutagen - handle with care and throw contaminated tips,
etc., into biohazard bags.
Step 4: Set up fluorometric plate reader following the manufacturer's
specifications.
Cell Viability Assay Procedure (process one plate at a time):
Step 1: Remove plate from incubator and remove media for
subsequent hormone analysis or extraction as described in
Subsection (h)(5), and add 300 |jL of PBS with Ca2+ and
Mg2+. Then rinse 1 time with 300 iliL of PBS with Ca2+ and
Mg2+. Note: Remove medium from not more than 4 wells at
a time and fill with PBS with Ca2+ and Mg2+ before removing
medium from second set of wells to prevent cells from
drying.
Step 2: QC plate only: Remove the PBS from wells A4-6 and B4-6
and add 300 |jL Methanol to these wells, and allow standing
at room temperature for 30 minutes.
Step 3: Remove the methanol and rinse those wells three times with
300 |jL PBS with Ca2+ and Mg2+. Rinse the balance of the
wells once with 300 iliL PBS with Ca2+ and Mg2+. Leave the
last 300 |jL rinse on the cells.
Step 4: Add 600 |jL of viability assay reagent to all wells.
IMPORTANT CONSIDERATION: Since the staining is
progressive up to at least 3 to 4 hours, it is important to
Page 32
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space the staining of each plate to the time it takes to read
the fluorescence.
Step 5: Incubate at room temperature for 1 hour (cover the plate with
lid to prevent evaporation).
Step 6: Scan the plate in the fluorometer using the above described
settings.
Step 7: Export/print data (check that values are appropriate,
otherwise adjust sensitivity and rescan).
(C) Data Analysis. Average the three measurements for calcein
AM and ethidium bromide. Divide the average calcein AM
fluorescence for each sample by its ethidium bromide
homodimer fluorescence to obtain a live to dead ratio. Graph
the average calcein AM fluorescence and standard deviation
for the negative control, solvent control, and each
concentration tested. Examine the calcein AM data visually. If
the blank has greater viability than the other treatments, the
solvent may be toxic to the cells. If viability is adversely
affected in the least dose and continues to decrease with
increasing concentration of the test substance, the test
substance may be toxic to the cells. In either of these cases,
the hormone data must be regarded with great suspicion. If
the solvent is toxic, try a different solvent or a lower
concentration of solvent. If the test substance is toxic
conclude that cytotoxicity is likely to preclude any other effects
at this test concentration.
Hormone Extraction and Analysis.
(i) Hormone Extraction from Medium. This procedure is followed if
the hormone measurement system may be affected by the
presence of culture medium. If it can be demonstrated that
hormone measurements can be made in the presence of medium,
this procedure may be omitted.
(A) Equipment, Materials, and Reagents.
Equipment: Vortex; Scintillation Counter (Beckman Coulter-
LS6500 Multi-purpose SC); Nitrogen Evaporator.
Materials: 100-1000, and 2-20 |jL Pipettes, Pipette Tips; Glass
Scintillation Vials (Research Products International
Corp. Cat#-211000); Glass Test Tubes (> 5 ml_) with
caps.
Page 33
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Reagents: Scintillation cocktail Bio-Safe II (Research Products
International Corp Cat# 111195); Ether, Anhydrous
(J.T. Baker Cat# 9244-22); Testosterone(1,2,6,7-
3H(N))-1mCi (PerkinElmer Cat# 370001).
(B) Method, Procedures and Requirements.
Step 1: Label two glass test tubes for each medium sample to be
extracted.
Step 2: Pipet 450 |jL medium into the test tube.
Step 3: Spike medium samples with 10 |jL of 3H-testosterone
(concentration = 0.0002 |jCi/|jL) to test for extraction
recoveries. (At this low concentration the radio-labeled
hormone spike will be detectable in a liquid scintillation
counter, but will not affect the end result of the hormone
concentration in the ELISA test).
Step 4: Briefly vortex medium samples after spiking.
Step 5: Add 10 |jL of the 3H-labeled hormone to a liquid scintillation
vial containing 4 ml_ of scintillation cocktail (this tube is used
as a reference for the calculation of CPM of the "CPM spike
tube" used later for the derivation of extraction efficiencies;.
Step 6: Bring all medium samples to 1 ml_ with nanopure water.
Step 7: Add 2.5-mL ether to each test tube, and cap. Be careful not to
remove test tube label in the process.
Step 8: Vortex each tube for a minimum of 1 minute to allow the water
and ether layers to mix.
Step 9: Allow the ether and water fractions to separate or centrifuge for
10 min at 2,000 rpm. Carefully collect the ether fraction
(supernatant) into a new test tube using a glass pipet without
disturbing water fraction.
Alternative method Prepare a dry ice-acetone bath in a
fume hood by placing several large chunks of dry ice in a
large glass beaker and filling with acetone. Hold plasma
samples in the dry ice bath for approximately 30 sec or until
water fraction is completely frozen. Then collect non-frozen
ether fraction as described above.
Page 34
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Step 10: Re-extract original water fraction by adding 2.5 mL ether to the
tube, cap, vortex, and centrifuge as above.
Step 11: Collect ether fraction into the same tube containing first ether
fraction.
Step 12: Wash ether with 1 mL nanopure water to remove hydrophilic
contaminants that may be present. Cap, vortex, and
centrifuge as above, and transfer ether to new glass vial.
Note: May not be necessary. Can be omitted if laboratory
can demonstrate that there is no difference in analytical
results between washed and non water treated samples.
Step 13: Evaporate the ether fractions to dryness under a stream of
nitrogen.
Step 14: If samples are to analyzed immediately, reconstitute in
250|j,L assay buffer that is provided with each ELISA kit and
vortex. If analyses are to be conducted at a later date, cap
vial and store dry at -80°C for up to 8 weeks.
Step 15: Remove 10 |jL of the assay buffer extract from each sample
and place in a liquid scintillation vial containing 4 mL of
cocktail.
Step 16: Test for extraction efficiency by running 3H-labled spike, 3H-
labled plasma and a blank sample (10 |jL of assay buffer)
extracts in the liquid scintillation counter.
Step 17: After reconstitution of the extract either use sample within 24
hrs in the ELISA (see next section) or freeze at -80°C until
further processing (maximum storage time of reconstituted
samples should not exceed 4 weeks).
Step 18: Calculate recoveries from scintillation counter readings
(CPM) as follows:
Equation:
(CPMsample - CPIVIBlank) X 25
% recovery = x 100
(CPMspike - CPM Blank )
(ii) Hormone Measurements Using Commercial Test Kits. Conduct
hormone analysis as specified in the manuals provided by the test
kit manufacturer. Most manufacturers have a unique procedure by
Page 35
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which the hormone analyses are run. Each sample should be run
at two dilutions each in triplicate to ensure that at least one reading
falls within the linear range of the standard curve of the assay.
Dilutions in the plates need to be adjusted such that expected
hormone concentrations for the solvent controls fall within the
center of the linear range of the standard curve of the individual
assay (Subection (g)(2)(i) and Figure 1). Non conformance with
this increases the risk of an over- or underestimation of the true
changes due to chemical exposure, and therefore, results in the
rejection of the data.
Final hormone concentrations are calculated as follows:
Equation:
Hormone concentration (per ml_) / recovery * dilution factor
Example:
Extracted:
Reconstituted in:
Dilution in Assay:
450 pL medium
250 pL assay buffer
1:10 (to bring the sample within the line range
of the standard curve)
150 pg/mL (already adjusted for final
concentration per ml_)
89 %
Hormone Concentration in Assay:
Recovery:
Final Hormone Cone = (150pg/ml_) - (0.89) x (250 pl_/450 pL) x10 = 936.3 pg/mL
(6) QC Parameters During Testing. In addition to meeting the criteria for the
QC plate, other quality criteria that pertain to variation between replicate
wells, replicate experiments, linearity and sensitivity of hormone
measurement systems, variability between replicate hormone measures of
the same sample, and % recovery of hormone spikes after extraction of
medium are provided in Table 7.
Table 7. Recommended Ranges and/or Variation (%) for H295R Assay Test Plate
Parameters.
Comparison Between
T
E2
Basal hormone production in SCs
Fold-greater than MDL
> 5-fold
> 2.5-fold
Exposure Experiments - Within Plate CV for
SCs (Replicate Wells)
Absolute Concentrations
< 30%
< 30%
Exposure Experiments - Between Plate CV for
Fold-Change
< 30%
< 30%
Page 36
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Comparison Between
T
E2
SCs (Replicate Experiments)
Hormone Measurement System -Sensitivity
Detectable fold-decrease
relative to SC
> 5-fold
> 2.5-fold
Hormone Measurement System - Replicate
Measure CV for SCs
Absolute Concentrations
< 25%
< 25%
Medium Extraction - Recovery of Internal 3H
Standard (If Applicable)
CPM
> 65% Nominal
(7) Data Reporting and Analysis.
(i) Data Processing and Statistics. To evaluate the relative
increase/decrease in chemically altered hormone production,
results need to be normalized to the mean solvent control (SC)
value for each assay {i.e., each 24-well plate of cells used to test a
given chemical), and results are expressed as changes relative to
the SC in each exposure plate. II data are expressed as mean +/-
standard deviation (SD). All doses that exhibit cytotoxicity greater
20% are omitted for further evaluation. Relative changes are
calculated as follows:
Equation:
Relative Change = (Hormone conc. in each well^Mean solvent hormone conc.)
If by visual inspection of the well and the digital photographs
described in Section 7 there appears to be an increase in cell
number, the apparent increase needs to be verisfied. If an increase
in cell numbers is verified, this should be stated in the data
evaluation report, and the hormone data normalized by dividing
hormone concentration by the relative change in the number of
viable cells.
Prior to conducting statistical analyses, the assumptions of
normality and variance homogeneity are evaluated. Normality are
evaluated using standard probability plots or any other appropriate
statistical method (e.g., Shapiro-Wilk's test). If the data are not
normally distributed, it is recommended that the data be
transformed to approximate a normal distribution. If the data are
normally distributed or approximate normal distribution, differences
between chemical treatments and solvent controls (SCs) are
analyzed using parametric test (e.g., Dunnett's Test). If data are
not normally distributed, an appropriate non-parametric test is used
Page 37
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(e.g., Kruskal Wallis, Steel's Many-one rank test). Differences are
considered significant at p < 0.05.
A summary of criteria for the evaluation of data has been provided
below (Table 8).
Table 8. Data Categorization Parameters for the Analysis of Results Obtained with the
H295R Steroidogenesis Assay
Parameter
Criterion
Statistical Significance
Response is considered to be statistically significant if the difference from
the Solvent Control has p < 0.05
Dose response
Data is expected to follow a dose response type profile at non-cytotoxic
doses, or doses that do not interfere with the hormone measurement assay
(note: response can be bi-phasic such as an increase at lower and a
decrease at higher doses, but changes randomly observed at only a few
concentration within the dose range are to excluded).
Interference with hormone
measurement assay
When marked interference of the chemical of interest with the hormone
measurement system utilized occurs (> 30% of hormone concentration
measured at the same dose at which interference occurred), this data is
omitted. In the case of weak to moderate interference (< 30% of hormone
concentration measured at the same dose at which interference occurred),
results may be corrected for the % interference.
Solubility
The results at concentrations for which cloudiness or a precipitate is
observed is not included.
Cell viability
Only non-cytotoxic concentration (> 80% cell viability) are included
(ii) Data Interpretation and Graphical Representation. Results are
provided both in graphical (bar graphs representing mean +/- 1*SD)
and tabular (LOEC, direction of effect, and strength of maximum
response that is part of the dose-response portion of the data)
formats (for example see Figure 1). The bars that correspond to
statistically significant results are designated with an asterisk. Data
assessment is only considered valid if it has been based on three
independently conducted experiments1. Furthermore, the
coefficient of variation for the LOECs (or NOECs if appropriate)
among the three experiments is expected not to exceed 30%. The
concentration range used in runs 2 and 3 may be tailored on the
basis of the results of run 1 to better define the dose response
range containing the LOEC.
1 An experiment or run is considered independent if it has been conducted at a different date and with
cells from a different culture plate.
Page 38
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Forskolin
100
II
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w
at
a>
£
CO
¦C
V
2
.0
10
£ 1
0.1
0.00010.001 0.01
0.1
pJVI
10 100
Letrozole
1.2
1
0.8
0.6
0.4
0.2
*
"txt
X
0.00010.001 0.01 0.1
jiM
10 100
Figure 3. Example of the graphical presentation and evaluation of data obtained during the conduct of
the H295R Steroidogenesis Assay. Asteriks indicate statistically significant differences from the solvent
control (p<0.05).
A chemical is judged to be positive if the fold induction is
statistically different from the solvent control following the
procedures described in Subsection (h)(5) at doses that fall within
the increasing or decreasing portion of the dose-response curve.
Statistically significant increases in fold induction indicate the
chemical is an inducer of one or more enzymes in the steroid
synthesis pathway. Statistically significant decreases in fold
induction indicate the chemical is an inhibitor of one or more
enzymes in the steroid synthesis pathway. Statistically significant
differences at concentrations that do not follow a dose-response
curve may be due to random effects; such results are considered to
be equivocal. As noted in Subsection (h)(5) results exceeding the
limits of solubility or at cytotoxic concentrations are not included in
interpreting results.
(iii) Data Reporting. The test report should include the following
information:
(A) Testing Facility:
~ Name of facility and location.
~ Name of study director, other personnel, and their study
responsibilities.
~ Dates the study began and ended.
(B) Test Substance, Reagents and Controls:
Page 39
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~ Identity (name/CAS No as appropriate), source, lot/batch
number, purity, supplier, and characterization of test
substance, reagents, and controls.
~ Physical nature and relevant physicochemical properties of
test substance.
~ Storage conditions and the method and frequency of
preparation of test substances, reagents and controls.
~ Stability of test substance.
Cells:
~ Source and type of cells.
~ Number of cell passages (cell passage identifier) of cells
used in test.
~ Description of procedures for maintenance of cell cultures.
Pre-test Requirements (if applicable):
~ Description and results of chemical hormone-assay
interference test.
~ Description and results of hormone extraction efficiency
measurements.
~ Standard and calibration curves for all analytical assays to
be conducted.
Test Conditions:
~ Composition of media.
~ Concentration of test chemical.
~ Cell density (estimated or measured cell concentrations at
24 hours and 48 hours).
~ Solubility of test chemical.
~ Incubation time and conditions.
Test Results:
~ Raw hormone concentration data for each well for controls
and test substances-each replicate measure in form of the
original data provided by the instrument utilized to measure
hormone production (e.g., OD, fluorescence units, CPM,
etc.).
~ Validation of normality or explanation of data transformation.
~ Mean responses +/-1 SD for each well measured.
Page 40
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~ Viability (cytotoxicity) data (If using the Live/Dead® stain,
include values for both live and dead fluorescence).
~ Check that QC requirements were met.
~ A bar graph showing relative (fold) change at each
concentration, SD and statistical significance as stated in
Subsection (h)(7)(ii).
(G) Data Interpretation, Discussion and Conclusions:
~ Apply the data interpretation procedure to the results and
discuss findings.
(8) Records, Documentation and QC Requirements. All QC requirements
and criteria are listed in Appendix 1. Data are expected to fall within the
acceptable ranges defined for each parameter. If these criteria are not met
it is recommended that the sample be re-analyzed or be dropped from the
data set (A notation is made on the spreadsheets that QC criteria were not
met).
Any anomalies and/or deviation from the specified method are
documented.
It is expected that all testing be conducted according to Good Laboratory
Practice standards, but if a lab is not GLP certified, GLPs should be
followed to the extent possible.
(i) References.
1. Hecker, M., Giesy, J.P., Jones, P.D., Higley, E.B., Newsted, J.L., Mehrle, P (2006).
Influence of cell passage and freeze/thaw events on basal production of 17(3-
estradiol and testosterone by H295R cells. Interim draft report by ENTRIX, Inc.,
submitted to US-EPA, Contract Code # GS-10F-0041L. September 2006.
2. Hecker, M., Giesy, J.P., Timm, G (2008). Multi-Laboratory Validation of the H295R
Steroidogenesis Assay to Identify Modulators of Testosterone and Estradiol
Production. Report by ENTRIX, Inc., submitted to the U.S.-Environmental
Protection Agency, Contract Code # GS-10F-0041L. February 2008.
3. Mosman T (1983). Rapid colorimetric assay for growth and survival: application to
proliferation and cytotoxicity. J. Immunol. Methods. 100, 45-50.
4. Shapiro R, and Page LB (1976). Interference by 2,3-dimercapto-1-propanol (BAL)
in agiotensim I radioimmunotassay. J Lab Clin Med. 1976 Aug (2): 22-31.
Page 41
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Appendix 1
- Freeze Down Identification
Symbol Freeze Down Number
a
A
Alpha
0
P
B
Beta
1
y
r
Gamma
2
5
A
Delta
3
8
E
Epsilon
4
c
Z
Zeta
5
T|
H
Eta
6
0
0
Theta
7
I
I
lota
8
K
K
Kappa
9
X
A
Lambda
10
n
M
Mu
11
V
N
Nu
12
S
Z
Xi
13
o
0
Omicron
14
71
n
Pi
15
P
p
Rho
16
a
z
Sigma
17
T
T
Tau
18
V
Y
Upsilon
19
$
O
Phi
20
X
X
Chi
21
V
P
Psi
22
(0
Q
Omega
23
A- 1
-------
Appendix 2 - Cell Culture Diagram (Example)
Note: For the specific purpose of the validation studies cells will be grown from an
original ATCC batch for five (5.0) passages, and then frozen down resulting (given that
cells from one culture plate were always split into three new plates as shown below) in a
maximum of 162 batches. One of these batches will then be started up in culture again
and grown for at least four (4) passages (passage #4.5 = plate ID# B/4.5/9/X) prior to
initiation of the exposure experiments. Only cells that have been frozen down one (1)
time (B generation) will be used for these experiments. The passage number is not to
exceed 10.
Name:
Date:
Project:
ATCC Lot Number:
Gas Phase
Liquid N2
From ATCC Lot#
Cell Line
Culture Start Date
ATCC Lot#
Freeze Down ID/Passage#.Passage # at which cells
were frozen down/Total# of Passages since starting
H295R
MM/DD/YY
xxxxxxx
A/3.0/03/A
H295R
MM/DD/YY
XXXXXXX
A/3.0/03/B
H295R
MM/DD/YY
XXXXXXX
A/4.0/04/A
H295R
MM/DD/YY
XXXXXXX
A/3.0/03/C
H295R
MM/DD/YY
XXXXXXX
A/4.0/04/C
H295R
MM/DD/YY
XXXXXXX
A/1.0/01/A
H295R
MM/DD/YY
XXXXXXX
A/2.0/02/A
H295R
MM/DD/YY
XXXXXXX
A/2.0/02/C
H295R
MM/DD/YY
XXXXXXX
A/5.0/05/A
H295R
MM/DD/YY
XXXXXXX
A/4.0/04/B
H295R
MM/DD/YY
XXXXXXX
A/2.0/02/B
Freeze ce s
gas phase
H295R
MM/DD/YY
XXXXXXX
B/3.5/08/A
H295R
MM/DD/YY
XXXXXXX
B/2.5/07/C
H295R
MM/DD/YY
XXXXXXX
B/1.5/06/A
H295R
MM/DD/YY
XXXXXXX
B/2.5/07/A
H295R
MM/DD/YY
XXXXXXX
B/2.5/07/B
A - 2
------- |